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PubMed1994

Compartmentalization of SHC, GRB2 and mSOS, and hyperphosphorylation of Raf-1 by EGF but not insulin in liver parenchyma.

Rat liver parenchyma harbors equal numbers of epidermal growth factor (EGF) and insulin receptors. Following administration of a saturating dose of EGF (10 micrograms/100 g body weight), there was a rapid (t1/2 approximately 1.1 min) internalization of receptor coincident with its tyrosine phosphorylation at residue 1173 and receptor recruitment of the adaptor protein SHC, its tyrosine phosphorylation and its association with GRB2 and the Ras guanine nucleotide exchange factor, mSOS, largely in endosomes. This led to a cytosolic pool of a complex of tyrosine-phosphorylated SHC, GRB2 and mSOS. It was demonstrated that these constituents were linked to Ras activation by the characteristic decrease in Raf-1 mobility on SDS-PAGE, which was maintained for 60 min after a single bolus of administered EGF. While insulin administration (15 micrograms/100 g body weight) led to insulin receptor beta-subunit tyrosine phosphorylation and internalization, there was little detectable tyrosine phosphorylation of SHC, recruitment of GRB2, association of a complex with mSOS or any detectable change in the mobility of Raf-1. Therefore, in normal physiological target cells in vivo, distinct signaling pathways are realized after EGF or insulin receptor activation, with regulation of this specificity most probably occurring at the locus of the endosome.

Adaptor Proteins, Signal Transducing↗

PubMed2002

The polarized epithelia-specific mu 1B-adaptin complements mu 1A-deficiency in fibroblasts.

The heterotetrameric AP-1A adaptor complex of clathrin-coated vesicles is ubiquitously expressed. The mu 1-adaptin subunit of the complex exists as the ubiquitous mu 1A and the polarized epithelia-specific mu 1B, which are 80% identical. In polarized epithelia, mu 1B is incorporated into the AP-1B complex, which is required for basolateral plasma membrane sorting of the low-density lipoprotein receptor. Binding of AP-1B to subdomains of the trans-Golgi network (TGN) appears to be part of the mechanism by which protein sorting is mediated. We expressed mu 1B in mu 1A-deficient fibroblasts to test for mu 1B function in non-polarized cells. AP-1B complexes were formed and bound to the TGN and to endosomes. Moreover, AP-1B restored the AP-1A-dependent sorting of mannose 6-phosphate receptors between endosomes and the TGN. This demonstrates that mu 1A and mu 1B do have overlapping sorting functions and indicates that AP-1A and AP-1B mediate protein sorting along parallel pathways between the TGN and endosomes in polarized epithelia.

Adaptor Protein Complex mu Subunits↗

PubMed2004

Afadin- and alpha-actinin-binding protein ADIP directly binds beta'-COP, a subunit of the coatomer complex.

Afadin DIL domain-interacting protein (ADIP) is a novel protein that binds both afadin and alpha-actinin and localizes at adherens junctions, which are formed by nectins and cadherins, cell-cell adhesion molecules. Afadin is an actin filament (F-actin)-binding protein which connects nectins to the actin cytoskeleton. alpha-Actinin is another F-actin-binding protein that is indirectly associated with cadherins through the catenin complex. ADIP is at least partly involved in the physical association of nectins and cadherins. We show here that ADIP furthermore binds beta'-COP, a subunit of the coatomer complex. ADIP co-localizes with beta'-COP at the Golgi complex in Madin Darby canine kidney and normal rat kidney cells. These results suggest that ADIP is involved in vesicle trafficking from the Golgi to the endoplasmic reticulum and through the Golgi complex by interacting with the coatomer complex.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Alternate paths from epidermal growth factor receptor to Akt in malignant versus nontransformed lung epithelial cells: ErbB3 versus Gab1.

In many human lung adenocarcinoma cell lines, a pathway involving epidermal growth factor receptor (EGFR), ErbB2 and ErbB3 receptors, phosphatidyl inositol 3-kinase (PI3K), Akt, glycogen synthase kinase 3-beta (GSK3-beta), and cyclin D1 controls cell growth, survival, and invasiveness. We have investigated this pathway in paired transformed/nontransformed cell lines from murine peripheral lung epithelium, E9/E10 and A5/C10. The E9 and A5 carcinoma lines expressed ErbB3 and transforming growth factor-alpha (TGF-alpha) and responded to TGF-alpha stimulation with protein complex formation including the p85 regulatory subunit of PI3K, activation of Akt, phosphorylation of GSK3-beta, and increased cyclin D1 protein and the cell cycle. ErbB3 and TGF-alpha were not detected in the nontransformed E10 and C10 cell lines. Nevertheless, exposure of E10 or C10 cells to TGF-alpha activated PI3K and Akt and increased cyclin D1 and cell growth. The effector pathway from the EGFR to PI3K in these nontransformed cells included the adaptor Grb2, the docking protein Gab1, and the phosphatase Shp2. Gab1 was highly expressed in E10 and C10 cells but not in the malignant E9 and A5 sister lines. Complexes of EGFR/Grb2/Gab1/Shp2 after TGF-alpha stimulation were prominent only in E10 and C10 cells. Thus, alternate pathways downstream of EGFR regulate mitosis in these paired malignant versus nontransformed lung cell lines.

Adaptor Proteins, Signal Transducing↗

PubMed1995

Previously identified protein of uncertain function is karyopherin alpha and together with karyopherin beta docks import substrate at nuclear pore complexes.

Previously, we had purified a cytosolic protein complex, termed karyopherin, that functions in docking import substrate at the nuclear envelope in digitonin-permeabilized cells and also had molecularly cloned and sequenced its 97-kDa beta subunit. We now report that the karyopherin alpha subunit is the previously identified protein NPI-1/SRP-1 of hitherto uncertain function. Using purified recombinant karyopherin alpha or beta subunit, we showed that neither karyopherin alpha nor karyopherin beta alone was sufficient for docking of import substrate at the nuclear envelope. Docking occurred only when both subunits were present. Moreover, docking of import substrate by the two recombinant karyopherin subunits was productive, as it led to nuclear internalization of the docked substrate in the presence of additional, previously characterized cytosolic factors. In a binding assay using immobilized karyopherin alpha and beta subunits and import substrate as a ligand, we found that only karyopherin alpha bound ligand. We suggest that karyopherin beta functions as an adaptor that binds both to karyopherin alpha and to any of a large number of docking sites that are represented by a repetitive peptide motif containing nucleoporins on both the cytoplasmic and nucleoplasmic side of the nuclear pore complex (NPC), bidirectionally ferrying a complex of karyopherin alpha-substrate across the NPC.

Animals↗

PubMed2004

Dual engagement regulation of protein interactions with the AP-2 adaptor alpha appendage.

Clathrin-mediated endocytosis depends upon the coordinated assembly of a large number of discrete clathrin coat components to couple cargo selection with rapid internalization from the cell surface. Accordingly, the heterotetrameric AP-2 adaptor complex binds not only to clathrin and select cargo molecules, but also to an extensive family of endocytic accessory factors and alternate sorting adaptors. Physical associations between accessory proteins and AP-2 occur primarily through DP(F/W) or FXDXF motifs, which engage an interaction surface positioned on the C-terminal platform subdomain of the independently folded alpha subunit appendage. Here, we find that the WXX(F/W)X(D/E) interaction motif found in several endocytic proteins, including synaptojanin 1, stonin 2, AAK1, GAK, and NECAP1, binds a second interaction site on the bilobal alpha appendage, located on the N-terminal beta sandwich subdomain. Both alpha appendage binding sites can be engaged synchronously, and our data reveal that varied assemblies of interaction motifs with different affinities for two sites upon the alpha appendage can provide a mechanism for temporal ordering of endocytic accessory proteins during clathrin-mediated endocytosis.

Adaptor Protein Complex 2↗

PubMed1997

Molecular characterization of the AP19 gene family in Arabidopsis thaliana: components of the Golgi AP-1 clathrin assembly protein complex.

AP19 is the smallest polypeptide component of AP-1, the clathrin associated protein complex found in clathrin-coated vesicles of the Golgi apparatus. Two genomic clones that encode homologues of AP19 were isolated from Arabidopsis thaliana (AAP19-1 and AAP19-2). Analysis of their nucleotide sequences predict proteins of 162 and 163 amino acids with mr of 18,913 and 18,758 respectively. Amino acid sequence comparisons with mammalian, yeast and plant clathrin associated sequences indicates that the Arabidopsis genes encode polypeptides that are more closely related to the AP19 proteins associated with clathrin-coated Golgi vesicles than to AP17, which is part of the AP-2 complex of endocytic clathrin-coated pits. Ribonuclease protection assays showed that both genes are expressed in all Arabidopsis tissues throughout development. Constitutive transcription of AAP19-1 was confirmed in transgenic Arabidopsis seedlings and plants containing an AAP19-1 promoter::beta-glucuronidase (GUS) fusion by ribonuclease protection assays and GUS histochemical staining.

Adaptor Protein Complex 1↗

PubMed1998

Dileucine-based sorting signals bind to the beta chain of AP-1 at a site distinct and regulated differently from the tyrosine-based motif-binding site.

In previous work, we showed that peptides from endocytosed proteins containing the tyrosine YXXphi sorting motif are recognized by the mu 2 subunit of AP-2, the plasma membrane clathrin adaptor protein complex. This interaction is activated by phosphoinositide lipids that are phosphorylated at the D-3 position of the inositol ring, and is also enhanced by the formation of clathrin-AP-2 coats. Here, we describe the detection of a specific interaction between peptides containing a second sorting motif, the dileucine motif, and AP-1, the clathrin adaptor complex responsible for sorting proteins at the trans-Golgi network (TGN). Surprisingly, the site of dileucine binding is the beta1 subunit, not mu 1. A YXXphi-containing peptide from a protein trafficked within the TGN does bind to mu 1, however. Phosphatidylinositol 3,4-diphosphate and 3,4, 5-triphosphate did not activate the interaction between dileucine-containing peptides and AP-1 but instead inhibited it, and clathrin-AP-1 coat formation did not alter the interaction. Thus, there are at least two physically separate binding sites for sorting signals on APs, which are also regulated independently.

Amino Acid Sequence↗

PubMed2004

Identification of a novel 14-3-3zeta binding site within the cytoplasmic tail of platelet glycoprotein Ibalpha.

The glycoprotein Ib-V-IX (GPIb-V-IX) complex interacts with subendothelial von Willebrand factor (VWF) to ensure recruitment of platelets at sites of vascular injury, a process that culminates in integrin alpha(IIb)beta(3)-dependent stable adhesion and spreading. Interaction of the 14-3-3zeta adaptor protein with the C-terminal 606-610 phosphoserine motif of the GPIbalpha subunit has been implicated in the control of alpha(IIb)beta(3) activation and cell spreading. In this study, we have examined potentially novel 14-3-3zeta binding sites by expressing mutant forms of GPIbalpha in Chinese-hamster-ovary (CHO) cells. Analysis of a series of neighboring 11-12 residue deletions identified a critical role for the 580-LVAGRRPSALS-590 sequence in promoting GPIbalpha-14-3-3zeta interaction. Development of a phosphospecific antibody demonstrated high levels of phosphorylation of the Ser587 and Ser590 residues in resting platelets (which became dephosphorylated during platelet spreading on VWF), and peptides containing these phosphorylated residues effectively displaced 14-3-3zeta from GPIbalpha. Analysis of single and double alanine substitutions of Ser587 and Ser590 demonstrated a major role for these residues in promoting GPIbalpha-14-3-3zeta binding. Moreover, these cell lines exhibited a defect in cell spreading on immobilized VWF. These studies demonstrate the existence of a second major 14-3-3zeta binding site within the cytoplasmic tail of GPIbalpha that has an important functional role in regulating integrin-dependent cell spreading.

14-3-3 Proteins↗

PubMed1996

Architecture of coatomer: molecular characterization of delta-COP and protein interactions within the complex.

Coatomer is a cytosolic protein complex that forms the coat of COP I-coated transport vesicles. In our attempt to analyze the physical and functional interactions between its seven subunits (coat proteins, [COPs] alpha-zeta), we engaged in a program to clone and characterize the individual coatomer subunits. We have now cloned, sequenced, and overexpressed bovine alpha-COP, the 135-kD subunit of coatomer as well as delta-COP, the 57-kD subunit and have identified a yeast homolog of delta-COP by cDNA sequence comparison and by NH2-terminal peptide sequencing. delta-COP shows homologies to subunits of the clathrin adaptor complexes AP1 and AP2. We show that in Golgi-enriched membrane fractions, the protein is predominantly found in COP I-coated transport vesicles and in the budding regions of the Golgi membranes. A knock-out of the delta-COP gene in yeast is lethal. Immunoprecipitation, as well as analysis exploiting the two-hybrid system in a complete COP screen, showed physical interactions between alpha- and epsilon-COPs and between beta- and delta-COPs. Moreover, the two-hybrid system indicates interactions between gamma- and zeta-COPs as well as between alpha- and beta' COPs. We propose that these interactions reflect in vivo associations of those subunits and thus play a functional role in the assembly of coatomer and/or serve to maintain the molecular architecture of the complex.

Amino Acid Sequence↗

PubMed1997

Genetic analysis of stomatogastric nervous system development in Drosophila using enhancer trap lines.

The stomatogastric nervous system (SNS) of Drosophila melanogaster is a small, simply organized neural circuitry which innervates the anterior enteric system. It is responsible for regulating the passage of food through the pharynx and esophagus and into the midgut. Here we show that the development of the SNS is amenable to genetic dissection. We screened lines from a P-element mutagenesis, selecting those with lacZ reporter gene expression and/or a phenotype in the SNS, associated glia, and garland cells. We report a collection of expression patterns and mutant phenotypes among lines found to have a mutation in genes required for the establishment of the larval SNS. Our results indicate that SNS development depends on pattern organizer genes including components of the Ras/Raf pathway.

Adaptor Protein Complex alpha Subunits↗

PubMed1999

Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes.

The Nef protein of primate lentiviruses downregulates the cell surface expression of CD4 through a two-step process. First, Nef connects the cytoplasmic tail of CD4 with adaptor protein complexes (AP), thereby inducing the formation of CD4-specific clathrin-coated pits that rapidly endocytose the viral receptor. Second, Nef targets internalized CD4 molecules for degradation. Here we show that Nef accomplishes this second task by acting as a connector between CD4 and the beta subunit of COPI coatomers in endosomes. A sequence encompassing a critical acidic dipeptide, located nearby but distinct from the AP-binding determinant of HIV-1 Nef, is responsible for beta-COP recruitment and for routing to lysosomes. A novel class of endosomal sorting motif, based on acidic residues, is thus revealed, and beta-COP is identified as its downstream partner.

Amino Acid Sequence↗

PubMed2004

Direct interaction between Smad3, APC10, CDH1 and HEF1 in proteasomal degradation of HEF1.

BACKGROUND: The Transforming Growth Factor-beta (TGF-beta) regulates myriad cellular events by signaling through members of the Smad family signal transducers. As a key signal transducer of TGF-beta, Smad3 exhibits the property of receptor-activated transcriptional modulator and also the novel ability of regulating the proteasomal degradation of two Smad3 interacting proteins, SnoN and HEF1. It has been shown that Smad3 recruits two types of Ub E3 ligases, Smurf2 and the Anaphase Promoting Complex (APC), to mediate SnoN ubiquitination, thereby enhancing SnoN degradation. The molecular mechanisms underlying Smad3-regulated HEF1 degradation are not well understood. Furthermore, it is not clear how Smad3 recruits the APC complex. RESULTS: We detected physical interaction between Smad3 and an APC component APC10, as well as the interaction between HEF1 and CDH1, which is the substrate-interacting component within APC. Detailed domain mapping studies revealed distinct subdomains within the MH2 domain of Smad3 for binding to APC10 and HEF1 and suggests the formation of a complex of these four proteins (Smad3, HEF1, APC10 and CDH1). In addition, the protein levels of HEF1 are subjected to the regulation of overexpressed APC10 and CDH1. CONCLUSIONS: Our data suggests that Smad3 may recruit the APC complex via a direct interaction with the APC subunit APC10 to regulate the ubiquitination and degradation of its interactor HEF1, which is recognized as an ubiquitination substrate by the CDH1 subunit of the APC complex.

Activin Receptors, Type I↗

PubMed2006

Conserved oligomeric Golgi complex subunit 1 deficiency reveals a previously uncharacterized congenital disorder of glycosylation type II.

The conserved oligomeric Golgi (COG) complex is a heterooctameric complex that regulates intraGolgi trafficking and the integrity of the Golgi compartment in eukaryotic cells. Here, we describe a patient with a mild form of congenital disorder of glycosylation type II (CDG-II) that is caused by a deficiency in the Cog1 subunit of the complex. This patient has a defect in both N- and O-glycosylation. Mass spectrometric analysis of the structures of the N-linked glycans released from glycoproteins from the patient's serum revealed a reduction in sialic acid and galactose residues. Peanut agglutinin (PNA) lectin staining revealed a decrease in sialic acids on core 1 mucin type O-glycans, indicating a combined defect in N- and O-glycosylation. Sequence analysis of the COG1 cDNA and gene identified a homozygous insertion of a single nucleotide (2659-2660insC), which is predicted to lead to a premature translation stop and truncation of the C terminus of the Cog1 protein by 80 amino acids. This mutation destabilizes several other COG subunits and alters their subcellular localization and hence the overall integrity of the COG complex. This results in reduced levels and/or altered Golgi localization of alpha-mannosidase II and beta-1,4 galactosyltransferase I, which links it to the glycosylation deficiency. Transfection of primary fibroblasts of this patient with the full length hemagglutinin-tagged Cog1 indeed restored beta-1,4 galactosyltransferase Golgi localization. We propose naming this disorder CDG-II/Cog1, or CDG-II caused by Cog1 deficiency.

Adaptor Proteins, Vesicular Transport↗

PubMed2001

Nef-induced CD4 downregulation: a diacidic sequence in human immunodeficiency virus type 1 Nef does not function as a protein sorting motif through direct binding to beta-COP.

The Nef protein from the human immunodeficiency virus (HIV) induces CD4 cell surface downregulation by interfering with the endocytic machinery. It has been recently proposed that binding of HIV type 1 Nef to the beta subunit of COPI coatomers participated in the Nef-induced CD4 downregulation through recognition of a novel diacidic motif found in the C-terminal disordered loop of Nef (V. Piguet, F. Gu, M. Foti, N. Demaurex, J. Gruenberg, J. L. Carpentier, and D. Trono, Cell 97:63-73, 1999). We have mutated the glutamate residues which formed this motif in order to document this observation. Surprisingly, mutation of the diacidic sequence of Nef did not significantly affect its ability (i) to interact with beta-COP, (ii) to downregulate CD4 cell surface expression, and (iii) to address an integral resident membrane protein containing Nef as the cytoplasmic domain to the endocytic pathway. Our results indicate that these acidic residues are not involved in the connection of Nef with the endocytic machinery through binding to beta-COP. Additional studies are thus required to characterize the residues of Nef involved in the binding to beta-COP and to evaluate the contribution of this interaction to the Nef-induced perturbations of membrane trafficking.

Adaptor Protein Complex gamma Subunits↗

PubMed2004

The structure of the MAPK scaffold, MP1, bound to its partner, p14. A complex with a critical role in endosomal map kinase signaling.

Scaffold proteins of the mitogen-activated protein kinase (MAPK) pathway have been proposed to form an active signaling module and enhance the specificity of the transduced signal. Here, we report a 2-A resolution structure of the MAPK scaffold protein MP1 in a complex with its partner protein, p14, that localizes the complex to late endosomes. The structures of these two proteins are remarkably similar, with a five-stranded beta-sheet flanked on either side by a total of three helices. The proteins form a heterodimer in solution and interact mainly through the edge beta-strand in each protein to generate a 10-stranded beta-sheet core. Both proteins also share structural similarity with the amino-terminal regulatory domains of the membrane trafficking proteins, sec22b and Ykt6p, as well as with sedlin (a component of a Golgi-associated membrane-trafficking complex) and the sigma2 and amino-terminal portion of the mu2 subunits of the clathrin adaptor complex AP2. Because neither MP1 nor p14 have been implicated in membrane traffic, we propose that the similar protein folds allow these relatively small proteins to be involved in multiple and simultaneous protein-protein interactions. Mapping of highly conserved, surface-exposed residues on MP1 and p14 provided insight into the potential sites of binding of the signaling kinases MEK1 and ERK1 to this complex, as well as the areas potentially involved in other protein-protein interactions.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Listeriolysin O secreted by Listeria monocytogenes induces NF-kappaB signalling by activating the IkappaB kinase complex.

Listeriolysin O (LLO) is a pore-forming cytolysin secreted by the pathogen Listeria monocytogenes and is required for its intracellular survival. We recently demonstrated that in endothelial cells, LLO activates the NF-kappaB signalling pathway. In this work, we studied the LLO-induced molecular cascade of NF-kappaB activation with a cellular model extensively used to analyse the signalling pathway of NF-kappaB activation, i.e. the human embryonic kidney HEK-293 cell line and its derivatives (transfectants or mutants). When the stably transfected derivative HEK-293 cells expressing IL-1RI were exposed to LLO, a strong NF-kappaB activation was detected, contrasting with other cell lines (HEK-293 wild type, HEK-293.T and COS) expressing a very low level of IL-1RI. Although a delayed kinetics of LLO-dependent NF-kappaB activation suggests an autocrine or paracrine IL-1-dependent pathway, we found that LLO-dependent NF-kappaB activation did not require the IL-1 protein synthesis nor the interaction with the IL-1RI specific receptor. Herein, we demonstrated that LLO-dependent NF-kappaB activation requires the activation of the IkappaB kinase beta (IKKbeta) subunit of IKK complex to phosphorylate and degrade cytoplasmic IkappaBalpha, a natural inhibitor of NF-kappaB. The activation induced by LLO does not require the adapters MyD88 and IL-1R-associated kinase (IRAK). We suggested that LLO induces a distinct signalling pathway from that of IL-1 and its receptor.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Endocytosis of the viral chemokine receptor US28 does not require beta-arrestins but is dependent on the clathrin-mediated pathway.

Arrestins bind phosphorylated G-protein coupled-receptors (GPCR) and inhibit agonist-induced signal transduction by uncoupling the receptors from their cognate G-proteins. beta-arrestins also act as adaptors that target GPCR to endocytic clathrin-coated vesicles. Unlike cellular GPCRs, the human cytomegalovirus GPCRs and chemokine receptor, US28, shows constitutive signal transduction activity and undergoes constitutive endocytosis. To determine the role of beta-arrestins in US28 trafficking, we used embryonic fibroblasts derived from beta-arrestin knockout mice. In these cells, the internalization of transfected beta2-adrenergic receptor and of the cellular chemokine receptor CCR5 was impaired. By contrast, US28 distribution was unaffected, and US28-mediated RANTES internalization was similar in normal and knockout cell lines. To investigate whether a clathrin-mediated pathway is involved in US28 endocytosis, we developed small interfering RNA against the micro2-adaptin subunit of the AP-2 adaptor complex. In cells transfected with micro2 small interfering RNA transferrin endocytosis was severely inhibited. Antibody-feeding experiments and biochemical analysis showed that US28 internalization was also inhibited. Together, these data indicate that US28 endocytosis occurs via a clathrin-mediated mechanism but is independent of beta-arrestins.

Animals↗